44 HLA TYPING AND TRANSPLANT IMMUNOLOGY

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Rajeswari S Moderator : Dr BIMAN SaIKIA:

Rajeswari S Moderator : Dr BIMAN SaIKIA HLA TYPING AND TRANSPLANT IMMUNOLOGY

What is an MHC ?:

What is an MHC ? The Major Histocompatibility Complex (MHC) is a large multigene family whose products are associated with the intercellular recognition and with self or non-self discrimination. The MHC is critical for immunological specificity, histocompatibility and susceptibilty for autoimmune diseases. In humans, the ~ 4 Mb (4,000,000bp) MHC region on Chromosome 6 contains about 140 genes , more than 20% of which have functions in immunity. Shows extensive conservation with the MHC of other other mammals (in mice MHC is termed H-2 complex), which helps in understanding MHC

Discovery of MHC:

Discovery of MHC The MHC was discovered as the genetic locus whose products were responsible for rapid rejection of tissue graft exchanged between inbred strains of mice. The particular locus that was identified in mice by Snell's group was linked to a gene on chromosome 17 encoding a polymorphic blood group antigen called antigen II, and therefore this region was named histocompatibility-2 or, simply, H-2. Genes that determine tissue compatibility between individuals- histocompatibility genes George Snell was awarded the Nobel Prize in 1980

Genome of HLA::

Genome of HLA: 20% genes are functional The most gene dense region 20 HLA genes;112 non HLA genes

Properties of MHC Molecules ::

Properties of MHC Molecules : Each MHC molecule consists of an extracellular peptide-binding cleft a pair of immunoglobulin ( Ig )-like domains anchored to the cell by transmembrane and cytoplasmic domains

Properties of MHC Molecules::

Properties of MHC Molecules: The polymorphic amino acid residues of MHC molecules are located in and adjacent to the peptide-binding cleft The nonpolymorphic Ig -like domains of MHC molecules contain binding sites for the T cell molecules CD4 and CD8

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Feature Class I MHC Class II MHC Distribution On nucleated cells; maximum on B cells; very low levels in fibroblasts, liver cells, muscle cells, neurons; sperm cells at certain stages and cells of placenta lack macrophages, dendritic cells and B-cells, thymus epithelial cells, Langerhans cells. Polypeptide chains α (44-47 kD ) β 2 - Microglobulin (12 k D ) α (32-34 kD) β (29-32 kD) polymorphic residues & antigen binding site α1 and α2 domains α1 and β1 domains Binding site for T cell coreceptor α3 region binds CD8 β2 region binds CD4 Antigen processed Endogenous protein Both endogenous and exogenous

Properties of MHC and peptide interactions::

Properties of MHC and peptide interactions: MHC molecules have a broad specificity for peptide binding (the fine specificity rests with the TCR ). Each Class I or Class II molecule has a single peptide-binding cleft that can accommodate many different peptides The association of peptide and MHC groove is a saturable , low- affinity interaction, with a slow on rate and a very slow off rate. The peptide molecules (anchor residues or sequence motifs) bind to the specific pockets (complementary residue) in the binding cleft of the MHC isoform .

Peptide binding by class I and class II MHC molecules:

Peptide binding by class I and class II MHC molecules Feature Class I Class II Peptide-binding domain α 1 and α 2 α 1 and β 1 Nature of peptide-binding cleft Closed at both ends Open at both ends General size of bound peptides 8–10 amino acids 13–18 amino acids Peptide motifs involved in Anchor residues at both ends of the peptide ;generally hydrophobic carboxyl-terminal anchor Anchor residues distributed along binding to MHC molecule peptide Nature of bound peptide Extended structure in which both ends interact with MHC cleft but middle arches up away from MHC molecule Extended structure that is held at a constant elevation above the floor of MHC cleft.

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Class I Class II

Genetic characteristics of HLA :

Genetic characteristics of HLA

HLA INHERITANCE::

HLA INHERITANCE: They follow Mendelian inheritance Expression of MHC alleles is codominant , meaning that the protein products of both the alleles at a locus are expressed equally in the cell, and both gene products can present antigens to T cells. The particular combination of MHC alleles found on a single chromosome is known as an MHC haplotype .

HLA inheritance::

HLA inheritance: Assuming no recombination, 4 different haplotypes are possible in the offspring . The inheritance pattern iimportant for compatible donors for transplantation. The chance that two siblings will be genotypically HLA identical is 25%. The chance that any one patient with “n” siblings will have at least one HLA-identical sibling is 1 – (3/4) n .

HLA inheritance:

HLA inheritance Having two siblings provides, a priori, a 44% chance, and having three siblings provides a 58% chance that one sibling will be HLA identical. The probability will never be 100% for finding an HLA-identical sibling. Each time a new sibling is tested, that new sibling has (only) a 25% chance of being a match, no matter how many siblings have previously been tested.

HLA GENE DIVERSITY::

HLA GENE DIVERSITY: The MHC is polygenic : it contains several different MHC class I and MHC class II genes, so that every individual possesses a set of MHC molecules with different ranges of peptide-binding specificities. The MHC is highly polymorphic ; that is, there are multiple variants, or alleles, of each gene within the population as a whole. The MHC genes are, in fact, the most polymorphic genes known .

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MHC class I and class II are polygenic (several loci encoding products with essentially the same function) a chain a chain a chain b 2 microglobulin is not encoded in MHC

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MHC polymorphism in the peptide binding groove explain the preference of different MHC proteins for different sequence motifs. Polymorphisms in MHC show how TCRs recognize MHC- allele-specific epitopes for restricted recognition of antigens For MHC class I, a 2 and a 1 domains are polymorphic; b 2 microglobin and the a 3 domain are not polymorphic For MHC class II, b 1 domains are polymorphic; a 2 and b 2 domains are not polymorphic Allelic variation in MHC occurs at the peptide binding site and on the floor /sides of the groove

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FOR 2010

Mechanisms::

Mechanisms: Point mutation (replacement substitutions/ silent mutations) Gene conversion Tandem duplication

Why Polymorphism and Polygenism in MHC? :

Why Polymorphism and Polygenism in MHC? Polygeny , ensures that each individual produces a number of different MHC molecules The high polymorphism of the classical MHC genes ensures a diversity in MHC gene expression in the population as a whole. However, no matter how polymorphic a gene, no individual can express more than two alleles at a single gene locus.

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T-cells are specific for amino acid sequence and MHC molecule

MLR (Mixed Lymphocyte reaction)::

MLR (Mixed Lymphocyte reaction): Studies have shown that roughly 1-10% of all T cells in an individual will respond to stimulation by cells from another, unrelated, member of the same species. This type of T-cell response is called an alloreaction or alloreactivity , because it represents the recognition of allelic polymorphism in allogeneic MHC molecules as explained by Mixed lymphocyte reaction.

Linkage Disequilibrium::

Linkage Disequilibrium: Expected frequencies for HLA haplotypes are derived by multiplication of the frequencies of each allele In reality the haplotypes are overrepresented than expected, which can be explained by Linkage Disequilibrium Assuming random distribution of haplotypes in European population, calculated as follows

Crossing Over::

Crossing Over: HLA genes occasionally demonstrate chromosome crossover in which segments containing linked genetic material are exchanged between the two chromosomes during meiosis or gametogenesis h aplotypes in offspring. It is related to the physical distance between the genes and their resistance or susceptibility to recombination . Example, the HLA-A, HLA-B, and HLA-DR loci are close together, with 0.8% crossover between the A and B loci and 0.5% between the B and DR loci. Crossovers HLA-B /HLA-C loci or HLA-DR /HLA-DQ loci are extremely rare

HLA Nomenclature, Loci and Alleles :

HLA Nomenclature, Loci and Alleles HLA nomenclature established by the WHO nomenclature committee in 1967 adheres to the following format: 1.HLA antigen nomencltaure ( by serology) 2. HLA allele nomenclature (molecular technique) 25

SEROLOGICAL SPECIFICITIES :

SEROLOGICAL SPECIFICITIES Nomenclature : Name of HLA Molecule Order in which the serological specificity defined ( eg HLA – A9 ) Over the time old serological specificities were split ( eg HLA – A9 : A 23 , A24 ) The most narrow definition of the specificity : Subtypic or PRIVATE SPECIFICITY The broader shared specificities : Supertypic specificities SO A CELL THAT IS A9 + MUST ALSO BE POSITIVE FOR A23 & A24.

HLA ANTIGEN NOMENCLATURE::

HLA ANTIGEN NOMENCLATURE: Splits”: Refinement of serologic methods permitted antigens that were previously believed to represent a single specificity to be “split” into specificities that were serologically (and, later, genetically) distinct . Cross-Reactive Groups (CREGs): HLA antigens and antigen groups may have other epitopes in common. Antibodies that react with the shared determinants often cause cross-reactions in serologic testing . The collective term for such cross-reactivity is cross-reactive group.

Serology Based Typing::

Serology Based Typing: A panel of known anti HLA antibodies are incubated with viable lymphocytes of unknown HLA type The HLA type of the sample is determined from the pattern of cell killing ( cytotoxicity ) that results from the antigen antibody reactions Advantages: 1.Rapid 2. Assesses HLA cell surface expression Disadvantages: 1.Limited detection of HLA polymorphism and low resolution 2.Requires viable cells 3.Requires HLA cell surface expression

SCORING OF LYMPHOCYTOTOXICITY TEST::

SCORING OF LYMPHOCYTOTOXICITY TEST:

HLA ALLELIC NOMENCLATURE::

HLA ALLELIC NOMENCLATURE: HLA refers to the genetic complex The second part is the name of the specific locus 6 loci- A, B, C, DR, DQ, DP .An allele is defined as a unique nucleotide sequence for a gene as defined by the use of all of the digits in a current allele name.

MOLECULAR TYPING :PRINCIPLE :

MOLECULAR TYPING :PRINCIPLE Molecular HLA typing methods can detect any polymorphic nucleotide sequence Advantages: Allele level matching Minimum no of cells required no viable cells required Improved accuracy and specificity : Specific oligonucleotide probes and primers Flexibility: New reagents can be designed as new alleles are discovered

HLA resolution techniques- Venn diagram:

HLA resolution techniques- Venn diagram High-resolution: HLA typing defines the specific DNA sequence of the antigen binding site. Allelic resolution defines a single allele as defined by a unique DNA sequence for the HLA gene

METHODS : :

METHODS : Methods Clinical application Resolution SSP (PCR) Solid organ, related and unrelated HPC transplantation Serologic to allele level, higher resolution with large number of primers DNA sequencing Unrelated HPC transplantation, resolution of typing problems with other methods, characterization of new alleles Allele level SSOP hybridization Solid organ and HPC transplanta - tion Serologic to allele level

ROLE OF HLA::

ROLE OF HLA: Solid organ transplantation Allogenic Hematopoietic stem cell transplantation Alloimmunization in multiple transfused patients Platelet refractoriness Neonatal alloimmune thrombocytopenia TRALI FNHTR TAGVHD HLA and autoimmune disease

Immunologic Basis of Graft Rejection: :

Immunologic Basis of Graft Rejection: The degree of immune response to a graft varies with the type of graft. The following terms are used to denote different types of transplants: Autologous (self) e.g., BM, peripheral blood stem cells, skin, bone Syngeneic (identical twin) Allogeneic (another human except identical twin) Xenogeneic (one species to another)

HLA and Hematopoietic cell transplantation::

HLA and Hematopoietic cell transplantation:

GRAFT REJECTION:

GRAFT REJECTION Rejection is a complex process in which both cell-mediated immunity (cellular)and circulating antibodies ( humoral ) play a role. Cellular rejection : Acute and chronic Allorecognition occurs by 2 ways: Direct and indirect allorecognition

MECHANISMS OF ALLORECOGNITION::

MECHANISMS OF ALLORECOGNITION: DIRECT : T cells of the transplant recipient recognize allogeneic (donor) MHC molecules on the surface of APCs in the graft through the DONOR dendritic cells. CD8+ T cells recognize class I MHC molecules  active CTLs  kill the graft cells. CD4+ helper T cells recognize allogeneic class II molecules and proliferate and differentiate into T H 1 (and possibly T H 17) effector cells. Cytokines secreted by the activated CD4+ T cells trigger a delayed hypersensitivity reaction in the graft-----  increased vascular permeability and local accumulation of mononuclear cells (lymphocytes and macrophages), and graft injury

MECHANISM OF ALLORECOGNITION::

MECHANISM OF ALLORECOGNITION: INDIRECT: The recipient T lymphocytes recognize MHC antigens of the donor cells after they are presented by the recipient's own APCs. The principal mechanism  T-cell cytokine production and delayed hypersensitivity. CD8+ CTLs that may be generated by the indirect pathway cannot directly recognize or kill graft cells, because these CTLs recognize graft antigens presented by the host's APCs Direct pathway is the major pathway in acute cellular rejection and indirect pathway is important in chronic rejection. This separation is by no means absolute

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HUMORAL REJECTION : IN SENSITIZED PATIENTS (HYPERACUTE): Antibodies produced against alloantigens in the graft cause humoral rejection  occurs within minutes after transplantation. kidney rapidly becomes cyanotic Immunoglobulin /complement are deposited in the vessel wall  endothelial injury and fibrin-platelet thrombi. PREVIOUSLY NOT SENSITIZED (ACUTE): Antibodies cause complement-dependent cytotoxicity and ADCC causing rejection vasculitis and renal atrophy

Graft versus host disease - Transplantation of hematopoietic stem cells:

Graft versus host disease - Transplantation of hematopoietic stem cells Two problems that are unique to bone marrow transplantation are graft-versus-host (GVH) disease and immunodeficiency. GVHD is a complex disease resulting from donor T-cell recognition of a genetically disparate recipient that is unable to reject donor cells after allogeneic HSCT. Billingham criteria : Immuno -incompetent host Infusion of competent donor T-cells HLA disparity between host and donor

Risk Factors of GVHD :

Risk Factors of GVHD HLA disparity and unrelated mismatched donors Allo stem cell source (More with PBSC) Donor Age Immunosuppression Increased dose of TBI Intense conditioning regimen Multi transfused donor Multiparous female donor /Male recipient

Incidence ::

Incidence : The incidence of GVHD with HLA- nonidentical marrow donors who are related or in HLA-matched unrelated donors  rates of 70-90%. Chronic GVHD is observed in 33% of HLA-identical sibling transplantations, in 49% of HLA-identical related transplantations, in 64% of matched unrelated donor transplantations. The rate could be as high as 80% in 1-antigen HLA- nonidentical unrelated transplantations (Atkinson K. Chronic graft-versus-host disease. Bone Marrow Transplant . Feb 1990;5(2):69-82)

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Steps of GVHD

Stages of GVHD:

Stages of GVHD Hyperacute Day 0 – 7 Acute Day 7 – 100 Chronic Day 100 ≥

Acute GVHD:

Acute GVHD Acute GVH disease occurs within days to weeks after allogeneic bone marrow transplantation. Although any organ may be affected, the major clinical manifestations result from involvement of the immune system and epithelia of the skin, liver, and intestines . Although tissue injury may be severe, the affected tissues are usually not heavily infiltrated by lymphocytes. It is believed that in addition to direct cytotoxicity by CD8+ T cells, considerable damage is inflicted by cytokines released by the sensitized donor T cells.

Acute GVHD:

Acute GVHD • Dermal: Maculopapular Palms / Soles Pruritic ± Cheeks/ Ears/ Neck / Trunk Necrosis / Bullae • Hepatic : Hyperbilirubinemia • Gastrointestinal : Diarrhea Abdominal pain Nausea ,Vomiting

Grading of acute GVHD:

Grading of acute GVHD

Chronic GVHD:

Chronic GVHD Multi system disorder Clinical & pathological findings resemble autoimmune diseases

Pathological Classification:

Pathological Classification Limited chronic GvHD Localised skin Hepatic involvement May not always require treatment & has a favorable outcome Extensive Chronic GvHD Generalised skin involvement Hepatic dysfunction Aggressive hepatitis Ocular involvement Involvement of the salivary gland Fatal if not treated

Effects of Chronic GvHD:

Effects of Chronic GvHD Skin (95%) cases –increase in collagen deposits -atrophy of the dermis -development of sclerosis - hyperpigmentation -patchy permanent hair loss Esophagus : difficulty in swallowing Sinuses – (increase risk of gram + ve bacterial infections Lungs - bronchiolitis obliterans Small intestine ( diarrhoea , malabsorption ) Vaginal ( inflammation, stricture & stenosis ) Other systems: CNS, nephrotic syndrome, persistent cystitis) Copyright Marvelle Brown

GVL EFFECT :

GVL EFFECT

Minor Histocompatibility antigens::

Minor Histocompatibility antigens: Unless donor T cells are depleted from the stem-cell graft, GVHD also frequently occurs after HLA-matched stem-cell transplantation because of recognition of minor histocompatibility antigens Polymorphic peptides that are displayed by HLA molecules of recipient cells. Endogenous proteins in recipient cells that differ from those of the donor, because of genetic polymorphisms, can provide distinct HLA-binding peptides and serve as minor histocompatibility antigens for donor T cells. The identification of haematopoietic-restricted minor histocompatibility antigens such as HA-1, HA-2,HB-1 and BCL2A1 is crucial for combining targeted immunotherapy with allogeneic stem-cell transplantation.

PLS( Passenger lymphocyte syndrome): :

PLS( Passenger lymphocyte syndrome): Three different groups of ABO incompatibility  minor, major, and bidirectional ABO incompatibility. Major ABO-incompatible (e.g., A into O)  presence of preformed antidonor A/B Ab directed against donor ABO Ag expressed on transplanted cells. Recipients of minor ABO-incompatible transplantation (e.g., O into A) express ABO Ag that are not expressed in the donor and are at risk for graft-versus-host ( GvH ) reactions such as delayed hemolysis of recipient red blood cell (RBC) due to PLS.

PLS::

PLS: Bidirectional ABO incompatibility (e.g., A into B) represents a combination of major and minor ABO incompatibility and puts the recipient at risk for both host-versus-graft and GvH . Immunocompetent donor memory B lymphocytes produce antibodies in a secondary immune response against the recipient’s red cells. The massive red cells destruction is thought to be complement-mediated. Occurs in solid organ transplantation It usually occurs in 1–3 weeks posttransplant and resolves within 3 months posttransplant , and is a self-limited process.

Methods to Reduce the Immunogenicity of Allografts :

Methods to Reduce the Immunogenicity of Allografts ABO blood typing; Determination of HLA alleles expressed on donor and recipient cells, called tissue typing. The detection of preformed antibodies in the recipient that bind to antigens of an identified donor's leukocytes, called crossmatching . Use of A2 organs with low titre antibodies for group B and O recipients Rituximab , splenectomy , plasmapheresis with intravenous immune globulin.

PowerPoint Presentation:

Why is typing necessary ? MHC matching - profound influence on graft survival before modern immunosuppressive drugs. Clinical experience has shown that of all the class I and class II loci, matching only HLA-A, HLA-B, and HLA-DR is important for predicting outcome. HLA-C is not as polymorphic as HLA-A or HLA-B, and HLA-DR and HLA-DQ are in strong linkage disequilibrium, so matching at the DR locus often also matches at the DQ locus . DP typing is not in common use, and its importance is unknown.

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GRAFT SURVIVAL WITH HLA MISMATCH

Hematopoietic Progenitor Cell Transplants::

Hematopoietic Progenitor Cell Transplants: Candidate donors and recipients are typed for their HLA-A, -B, -C, -DR, and -DQ alleles and, in some cases, for their HLA-DP allees . Match at allele level Some transplant programs additionally attempt to match for HLA-DQ or -DP alleles, or both. Molecular HLA typing is performed on samples from both the donor and recipient for optimal assessment of Class I and Class II region compatibility.

CONT…:

CONT… Because two codominantly expressed alleles are inherited for each of these MHC genes, it is possible to have zero to six MHC antigen mismatches between the donor and recipient. Zero-antigen mismatches predict the best living donor graft survival, and one-antigen-matched grafts do slightly worse. The survival of grafts with two to six MHC mismatches all are significantly worse than zero-or one-antigen mismatches

Crossmatching:

Crossmatching A pre-transplant test Ensures absence of donor reactive antibodies Prevents hyperacute rejection Prevents accelerated antibody mediated rejection. Techniques: Serology ( CDC test ) Solid phase assays ( Flow cytometry or Microarray technology)

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